Improving Separations Throughput with Existing Instrumentation

As laboratories look for ways to save time and money, some enterprising scientists have devised a way to do both in their work. Lance Heinle and Gary Jenkins, drug-development scientists at Abbott Laboratories (Abbott Park, IL), have created a high-throughput LC–MS-MS system using instrumentation already in place in their laboratory. In an interview, Heinle explained the thinking behind the endeavor.

Your article in the October Current Trends in Mass Spectrometry (“Creating a High-Throughput LC–MS-MS System Using Common Components”) describes a system compiled from instruments from several manufacturers. What factors were involved in choosing this approach rather than using an “out-of-the-box” system from one manufacturer (for example, cost, improved flexibility, higher sensitivity, speed of deployment)?

The driving factor behind this work was to find a way to deliver decision-making ADME data to our discovery teams in a shorter timeframe, without affecting the quality of our work. The logical first step was to ensure that we were utilizing our existing instruments their fullest capacity. We knew that there was room to optimize our analytical processes further, but did not know to what extent. If our efforts on the existing systems did not produce the desired cycle time reductions, we would have evaluated out-of-the-box systems in more detail. Like most companies, capital purchases are carefully considered, so if one can reach a desired endpoint without having to buy additional equipment, the savings can be used toward other initiatives. Fortunately, we were able to achieve our goal with in-house components in this case.

You mention in your article that the costs of out-of-the-box systems can be quite high. How much pressure are pharmaceutical R&D labs under today to reduce instrumentation costs?

In regards to pharmaceutical R&D, I think a better way to view it is ensuring that any money spent is on the most critical initiatives. One needs to weigh the pros and cons of each purchase and determine how it will not only help achieve the goal at hand, but any future needs as well (can this piece of equipment be used by other groups, to improve other processes, to drastically reduce a bottleneck, and so forth). We must also consider if we do have existing equipment that can fit the need. Taking a step back to question whether any investment is sound is not only smart, but fiscally responsible. One important factor to keep in mind is that reducing costs should not come at a cost to quality. However, we are fortunate to have the support of management, within reason, to purchase the right instrumentation if the need is justified.

How much did you have to adapt your methods when using the system you put together?

In terms of sample extraction and solvents, our methods were not adapted very much. While we tested various parameters, we ended up selecting the same mobile phases and column stationary phase (C18) as our original method. We found that for our chemical space, these parameters work fairly universally. One modification was selecting a column particle diameter. We tested various particle diameters, but determined that 5 µm was the best option for this application.

The biggest changes were made to the hardware. As detailed in the article, the sample loops were removed to reduce some of the dead volume in the HPLC systems and some minor, but easy, modifications were made to how to autosampler retrieved the samples.

Your article mentions that you encountered problems using columns with smaller particle diameters. What additional sample preparation steps would be necessary to enable the use of columns with particles smaller than 5 µm?

I believe a filtration or solid-phase extraction (SPE) step would be an effective way to clean up the samples for smaller particle sized columns. These steps would yield a solution effectively free of any particulates that would otherwise rapidly clog the column. Another possibility would be to transfer the solution to a new plate after centrifugation, leaving the protein pellet behind, reducing the chance of particulate from the pellet entering into the system. Of course, both of these options come with additional potential sources of error (such as loss of sample volume, pipetting errors, and nonspecific binding), as well as added cost.

Another factor to consider is the number of injections for an application. In our experiment, we needed to reliably inject 2304 samples without stopping. With our standard “crash and shoot” plates, we had some success using smaller-particle-size columns, but it was not consistent. If the injection number were less, then using a smaller particle sized column may be a viable option.

Would the disadvantages of additional sample preparation outweigh the benefits of higher resolution and sensitivity provided by the smaller-particle-size columns?

I believe this question should be considered on a case-by-case basis. For our needs, the minimal loss of sensitivity outweighed the additional time and cost of additional sample clean up. We came to this decision for two reasons. First, the time required for additional sample prep could dramatically extend the overall cycle time of the assay. If a scientist is performing an assay on several hundred compounds each week by him- or herself, there may not be time that day for additional sample prep once the time for assay set-up, incubation, and take-down are factored in. This could result in the LC–MS analysis not being started until the next morning. Second, we were able to achieve a 94% analytical success rate with the larger particle size, which already exceeded our original goal of 90%. In addition, not all of the failures were due to a lack of sensitivity, but were largely due to a failure of the stationary phase to retain the compound.

However, if sensitivity is a limiting factor in the success of a particular assay, then using smaller-particle-size columns with additional sample preparation would be a potential solution. It should also be pointed out that another benefit to additional sample preparation would be extending the life of the column. This may be an important consideration if working with a particularly expensive column.